This proposal, responsive to PAR-16-121 (Early-Stage Preclinical Validation of Therapeutic Leads for Diseases of Interest to the NIDDK; R01 mechanism) seeks to define principles for molecular design of ultra-stable single-chain insulin (SCI) analogs for the treatment of diabetes mellitus (DM). Previously studied as basic probes of structure-activity relationships, such analogs exhibit remarkable thermal stability and promise to circumvent the costly global cold chain of insulin delivery, distribution, storage and use. We posit: Hypothesis. The SCI framework will provide a shared platform for development of optimized rapid-acting (prandial), long-acting (basal) and biphasic (pre-mixed) formulations. The proposed preclinical studies promise to enable 3 therapeutic advances: (i) ultra-stable heat-resistant rapid-acting, biphasic and prolonged-acting SCI formulations for general usage; and (ii) extension to implanted intraperitoneal insulin pumps (IIIPs) in closed-loop (?smart?) systems as this technology is currently limited by degradation of insulin at body temperature. Molecular design is based on recent advances in the structural understanding of the insulin receptor (IR) and its mode of hormone binding [Menting, J.G., et al. Nature (2013) 493, 241-5, and PNAS-Plus (2014) 111, E3395-404]. An interdisciplinary Approach is proposed by a team of senior investigators respectively experienced in each of the component technologies. The essential idea exploits a conformational switch between a closed state of the hormone (as in a vial, pen or pump reservoir) and an open state (as observed on binding to a model IR complex; ?micro-receptor?). An ideal therapeutic insulin analog would combine enhanced stability of the closed state with native competence for reorganization (induced fit) in the hormone-IR complex. Aims 1-3 build on our preliminary findings to prepare and characterize a logical series of SCIs designed to exhibit a variety of pharmacokinetic and pharmacodynamics properties (PK/PD) following SQ and IV injections (including clamp studies). The studies will explore biochemical and cellular mechanisms, including assessment of potential hepato- and tissue-specific metabolic selectivity. These Aims are organized by deliverable: Aim 1 focuses on a rapid-acting SCI; Aim 2 on a basal SCI; and Aim 3 on a biphasic thermally-stable SCI with prolonged shelf-life and of particular relevance to the developing world. Animal studies will employ rats (STZ and an obese insulin-resistant model), dogs and pigs. The application contains explicit milestones and timelines related to serial exits via University Technology Transfer, anticipated to enable preparation of respective IND applications to the FDA. The feasibility of our Aims is supportive by extensive preliminary results, including two team manuscripts focused on structure and function of SCIs in final review at the JBC (2017). We anticipate that these key articles will soon be published and available to the Study Section online.